Synthesis and in-vitro anti-proliferative with antimicrobial activity of new coumarin containing heterocycles hybrids

A series of new coumarin-N-heterocyclic hybrids, coumarin-quinolines 7a–e, coumarin-acridines 10b,c and coumarin-neocryptolepines 13b,c were synthesized and evaluated for their anticancer and antimicrobial activities. The structures of all synthesized hybrids were confirmed by FT-IR, 1H-NMR, 13C-NMR, and MS spectrometry. The anti-proliferative activity of hybrids 7a–e, 10c and 13c were bio-evaluated using MTT-assay against colon (CaCo-2), lung (A549), breast (MDA-MB-231), and hepatocellular carcinoma (HepG-2) human cancer cell lines using doxorubicin as a reference drug. The results demonstrated that, all hybrids displayed moderate to good anti-proliferative activity against the cell lines. The most active hybrids were 7a–d and 10c against CaCo-2 cancer cell line with IC50: 57.1, 52.78, 57.29, 51.95 and 56.74 µM, and selectivity index 1.38, 1.76, 2.6, 1.96 and 0.77; respectively. While, 7a,d were potent against A549 cancer cell line with IC50: 51.72, 54.8 µM and selectivity index 1.5, 0.67; respectively. Moreover, 7c showed the most potency against MDA-MB-231 cancer cell line with IC50: 50.96 µM and selectivity index 2.20. Interestingly, docking results revealed that binding energy of the current compounds showed marked affinity values ranging from -6.54 to -5.56 kcal with interactions with the reported key amino acid SER 79. Furthermore, the antimicrobial activity of the synthesized hybrids 7a–e, 10b,c, 13b and 13c were evaluated against Gram‐positive and Gram‐negative bacterial and fungal strains. The hybrids 10b, 13b, 10c, and 13c exhibited broad-spectrum antibacterial activity against E.coli, S. mutans, and S. aureus with MIC from 3.2 to 66 µM, this hybrids also displayed antifungal activity against C. albicans with MIC values ranging from 0.0011 to 29.5 µM. In-silico investigation of the pharmacokinetic properties indicated that tested hybrids had high GI absorption, low Blood Brain Barrier (BBB) permeability in addition to cell membrane penetrability.


Synthesis and in-vitro anti-proliferative with antimicrobial activity of new coumarin containing heterocycles hybrids
A series of new coumarin-N-heterocyclic hybrids, coumarin-quinolines 7a-e, coumarin-acridines 10b,c and coumarin-neocryptolepines 13b,c were synthesized and evaluated for their anticancer and antimicrobial activities.The structures of all synthesized hybrids were confirmed by FT-IR, 1 H-NMR, 13 C-NMR, and MS spectrometry.The anti-proliferative activity of hybrids 7a-e, 10c and 13c were bio-evaluated using MTT-assay against colon (CaCo-2), lung (A549), breast (MDA-MB-231), and hepatocellular carcinoma (HepG-2) human cancer cell lines using doxorubicin as a reference drug.The results demonstrated that, all hybrids displayed moderate to good anti-proliferative activity against the cell lines.The most active hybrids were 7a-d and 10c against CaCo-2 cancer cell line with IC 50 : 57.1, 52.78, 57.29, 51.95 and 56.74 µM, and selectivity index 1.38, 1.76, 2.6, 1.96 and 0.77; respectively.While, 7a,d were potent against A549 cancer cell line with IC 50 : 51.72, 54.8 µM and selectivity index 1.5, 0.67; respectively.Moreover, 7c showed the most potency against MDA-MB-231 cancer cell line with IC 50 : 50.96 µM and selectivity index 2.20.Interestingly, docking results revealed that binding energy of the current compounds showed marked affinity values ranging from -6.54 to -5.56 kcal with interactions with the reported key amino acid SER 79.Furthermore, the antimicrobial activity of the synthesized hybrids 7a-e, 10b,c, 13b and 13c were evaluated against Gram-positive and Gramnegative bacterial and fungal strains.The hybrids 10b, 13b, 10c, and 13c exhibited broad-spectrum antibacterial activity against E.coli, S. mutans, and S. aureus with MIC from 3.2 to 66 µM, this hybrids also displayed antifungal activity against C. albicans with MIC values ranging from 0.0011 to 29.5 µM.In-silico investigation of the pharmacokinetic properties indicated that tested hybrids had high GI absorption, low Blood Brain Barrier (BBB) permeability in addition to cell membrane penetrability.
In recent years, the number of people suffering from cancer and multi-resistant infections has increased, such that both diseases are already seen as current and future major causes of death.Moreover, chronic infections are one of the main causes of cancer, due to the instability in the immune system that allows cancer cells to proliferate.Likewise, the physical debility associated with cancer or with anticancer therapy itself often paves the way for opportunistic infections.Though advances in cancer therapy and diagnosis have considerably improved life expectancy 1 , the overall survival rate of patients remains poor 2 .Disseminated cancer at the time of diagnosis and acquisition of tumor resistance are two main reasons.The growing knowledge of the biochemical pathways

Cell lines and microbial strains
Microbial strains: Escherichia coli, pseudomonas aeruginosa, Staphylococcus aureus, Streptococcus mutans and Candida albicans were collected from the microbial culture collection of Biopharmaceutical Products Research Department, Genetic Engineering and Biotechnology Research Institute, SRTA-City, New Borg El-Arab city, Alexandria, Egypt.

Molecular docking
Molecular docking studies were effectuated using and visualized on Biovia DS-2021 online free software to examine its affinity to topoisomerase II(Topo-II).Topo-II protein (pdb: 3FOE) was downloaded from protein data bank 44 .Protein preparation was done by deleting water molecules, selecting the co-crystalized ligand to be used as a current selection for defining the active site of the Top II and create binding site sphere attributes which was then extended to cover the whole protein.The ligand was then deleted, polar hydrogen was added, and files are saved as optimized protein.Ligand optimization and docking was performed by selecting dock ligand in lib-dock to prepare the ligand automatically in the program using the input site sphere attributes which was created upon protein optimization and minimize energy for the macromolecules before docking.The selected minimization force field was CHARMm.

Instrumental characterization
1 H-NMR and 13 C-NMR spectroscopic analyses were carried out with Bruker Germany; 400 and 100 MHz; respectively.Reports of chemical shifts were made in parts per million (ppm) as it relates to the respective solvent (DMSO-d 6 ).Also, FT-IR spectra were performed with Alpha, Bruker Germany at faculty of Science, Zagazig University.Thermo-Scientific GCMS model ISQ, USA, was used for the mass-spectra, which were conducted on the direct inlet part; at the regional center for Mycology and Biotechnology (RCMB).Exploring the characteristic fragmentation using Electron Impact mode and expected molecular weight at 70 eV.Melting point was recorded using scientific melting point apparatus without correction.

General synthesis of coumarin-N-heterocyclic hybrids 7a-e, 10b,c and 13b,c.
Coumarin-3-carbonyl chloride 3 (0.30 g, 1.27 mmol), appropriate amines 6a-e, 9b,c and 12b,c (1.27 mmol) dissolved in CH 2 Cl 2 (2 mL), triethylamine (0.39 g, 3.81 mmol) was added dropwise with stirring at room temperature (25˚C).Progress of the reaction was observed by thin layer chromatography (TLC) till starting materials were consumed (12 h.).The reaction mixture was poured into ice/water, and extracted three times with CH 2 Cl 2, the organic layer was collected, dried followed by removing of the solvent residue using rotatory evaporator.The precipitated solid was filtered off, dried and recrystallized from ethanol to give pure 7a-e, 10b,c and 13b,c in good yields.

Safety assays and anti-proliferative activities of Coumarin hybrids
The safety assays of coumarin hybrids on noncancerous cell lines and their anticancer effects on cancerous cells were gauged utilizing MTT-assay (Promega) in accordance with the instruction protocol.Starting from 60 to 200 µM, serial dilutions of coumarin hybrids were prepared in sterile DMSO to be treated cell lines at final concentrations 30 to 100 µM.The treated cells were incubated for 2 days, and the cellular cytotoxicity was detected by quantifying the solubilized formazan in DMSO at 570 nm.The inhibition concentration of 50 (IC 50 ) was calculated from the cytotoxicity% curve using GraphPad prism 9.

Selectivity index of Coumarin hybrids
Cancer cell selectivity index of Coumarin hybrids samples were measured as explained by 45 , with a minor modification; (SI = IC 50 nc/IC 50 cc), where IC 50 nc refers to the value of IC 50 of the coumarin hybrids compounds on normal cells, while IC 50 cc refers to the IC 50 of the coumarin hybrids compounds on cancer cell line.

Antimicrobial activities of coumarin hybrid
The antimicrobial activity of coumarin hybrids samples were checked against different multiple drug resistant microorganisms (Escherichia coli, pseudomonas aeruginosa, Staphylococcus aureus and Streptococcus mutans and Candida albicans) using various concentrations (30 to 100 µM).An aliquot of 100.0 µl of each sample concentration was added to an equal volume of each microbial growth dilution (about 10 6 CFU/ml) and inoculated into 96 well plate.Furthermore, 100.0 µl of LB media was added to 100.0 µl of microbial growth to set as the negative control group.After that, the inoculated plates were incubated overnight at 37 °C then, the microbial turbidity was measured using automated ELIZA microplate reader (BINDER BIOTECK E LX 800) adjusted at 620 nm.The microbial inhibition percentage after treatments were quantified using the following equation: where, A: the treatment group absorbance, A1: the blank absorbance, and A0: the control group absorbance.The Minimal Inhibitory Concentrations (MIC) were estimated and expressed as the lowest concentration of the tested samples which resulted in microbial growth inhibition.

Ethical statement
All research studies followed the Helsinki World Medical Association's Declaration: Ethical Medical Research Principles Involving Human Subjects and were approved by the ethics committee at Menoufia University in Egypt, Faculty of Science.Where, no animal experiments were conducted in this research, while all used cell lines were obtained from ATCC with original and recognized serial number of each cell line as aforementioned in section of cell line and microbial strains.

Synthesis of coumarin-3-carbonyl chloride
The key intermediate acid chloride 3 was prepared in good yield as pale-yellow crystals according to published method 21,46,47 , by the reaction of coumarin-3-carboxylic acid 1 and thionyl chloride (SOCl 2 ) refluxed for 2 h as depicted in Scheme 1.

Synthesis of 4 bis-aminosubstituted heterocycles
The reaction of 4,7-dichloroquinoline 4 with hydrazine, aliphatic and aromatic diamines 5a-e in presence of triethyl amine as a base catalyst afforded 6a-e in good yields via nucleophilic aromatic substitution (S NAr ).The synthesized compounds showed analytical data consistent with previously published results 4,48 .
The synthetic pathway for formation of coumarin-quinoline hybrids 7a-e was achieved by the reaction of 3 with diamines 6a-e, in equimolar ratio in presence of triethyl amine as a base to afford the corresponding hybrids 7a-e, in good to excellent yields (80-92%) as given in Scheme 1.Additionally, the target hybrids 10b,c were synthesized by the condensation of 9-chloroacridine 8 with amines 5b, 5c afforded the 9b and 9c, according to the reported method. 49,50Further reactions of 9b,c with 3 in equimolar ratio in presence of excess of triethyl amine in good yield (77,82%) as depicted in Scheme 2.
Furthermore, the condensation of 11-chloroneocryptolpine 11 with 5b and 5c yielding 12b,c in good yield (79%) according to the reported methods 51,52 .While, the reaction of 12b, 12c with 3 in equimolar ratio in presence of excess of triethyl amine afforded the corresponding coumarin-neocryptolepine hybrids 13b,c in good yields (79,85%), as shown in scheme 2. The progress of reactions was easily monitored by TLC using benzene and ethanol as eluent mixture (3:1), till starting materials are consumed.NMR analysis of coumarin hybrids 1 H-NMR spectrum shows multiple δ 3.46 and 3.89 ppm which correspond to the aliphatic spacers in 7b.Moreover, the spectra showed multiple with δ: 1.93 ppm, broad multiple with δ: 3.46 and 3.64 ppm, that confirmed the presence of aliphatic spacer in 7c.On the other hand, spectra showed multiple with δ: Moreover, the spectra showed multiple with δ: 4.02 ppm which confirmed the presence of (Ph-CH 2 -Ph) for 7e, showed broad multiple with δ: 4.16, 4.05 for which confirmed the presence of (N-CH 3 ) for 13b,c.On the other hand, coumarin (CH Ar ) appear with δ: 8.

Safety assays and anticancer activities of coumarin hybrids compounds
The safety profiles on WISH cells and the anticancer potentialities of coumarin hybrids compounds against MDA-MB-231, CaCo-2, A549 and HepG-2 cell lines were measured utilizing MTT-assay (Fig. 2A,B).The concluded data indicated that the compounds 7c, 7d and 13c were the safest compounds on WISH cells with IC 50 values of 112.39, 138.39 and 95.88; respectively (Table 1).While, among the control samples, 1 and coumarin (Ct) samples were safer than the other coumarin hybrids compounds with IC 50 values of 333.57and 176.36 µM; respectively (Fig. 2A and Table 1).
On the other hand, the most active compounds showed different anti-proliferative profiles on different cancerous cell lines.For example, 7c and 7d were the most effective compounds against CaCO-2 cell line with IC 50 values of 57.29 and 51.59 µM, with anticancer selectivity index 1.9 and 2.6; respectively (Fig. 2A).On A549 cell lines, the treatments 7a, 7b and 13c were the most significant treatments with selectivity index of 1.5, 1.6 and 1.4; respectively (Table 2).Also, 7c was the most potent treatment against MDA-MB-231 cell line with SI 2.2   www.nature.com/scientificreports/(Fig. 2B).Furthermore, 7d was the most potent compound against HepG-2 cell line with IC 50 82.83µM and SI 1.67 (Fig. 2A).

Antimicrobial activities of coumarin hybrids
The antimicrobial potency of coumarin hybrids was tested against different multiple drug resistant strains using microplate assay method.The results indicated that all investigated hybrids showed positive effects against all investigated microbes.Furthermore, samples 10b and 10c considered as the most efficient treatment against p. aeruginosa with MIC value of 14.8 and 15.4 µM, respectively (Table 3).Also, against both C. albicans, S. aureus samples 10c and 13c were the most potent treatments with MIC values of 0.0011 and 0.0013 µM, and 3.2 and 7.2 µM, respectively (Fig. 3).Concerning the antimicrobial activity against E. Coli, 10b, 13b and 13c were the most potent compounds with MIC values of 9.5, 9.8 and 9.4, respectively (Fig. 4).Finally, 10c, 13c and 13b were the most effective against Strep.mutans with a MIC value of 5.2, 14.8 and 28.5μM, respectively (Table 3).

Molecular docking studies
The anticancer effect of coumarins is assumed to be attributed to a variety of mechanisms, such as regulation of the estrogen receptor, activation of cell death, blockage of the cell cycle, and inhibition of DNA-associated enzymes including telomerase and topoisomerase (TOP). 36,53,54A significant portion of fundamental cellular biology involves DNA topoisomerases, which are also molecular targets for several medications, including antibiotics, antibacterials, and anticancer medications.They work by preventing the topoisomerase molecule from relegating DNA strands upon cleavage, so changing it into an agent that damages DNA 55,56 .In the field of medicinal chemistry and drug development, hybrid molecules which are created by combining two or more pharmacophores are a relatively new idea that has gained a lot of attention lately.Therefore, hybridization of coumarin with other anticancer pharmacophores may provide novel bioactive candidates with potential activity as well as low cytotoxicity.8][59][60] .
In-silico screening of the quinoline based compounds revealed their promising affinity to Topoisomerase II which is consistent with studies done on similar compounds as acridine and neocryptolipines 61 .The proposed binding mode of the investigated compounds showed marked affinity values ranging from a maximum value of -6.5392 kcal/mol recorded by 7e and a minimum value of -5.5638 kcal/mol recorded by 7c.Docking results disclosed that the planar di or tri ring aromatic system showed pi-hydrophobic or π-π interactions mostly with the amino acids residues Asp78, Asp83, Ser79 while, DG1, DC4, and DG5 were the residues for interaction at the DNA-minor groove, (Fig. 5).These interaction are consistent with the reported interactions by the cocrystallized ligand "7-[(3R)-3-aminopyrrolidin-1-yl]-8-chloro-1-cyclopropyl-6-fluoro-4-oxo-1,4-dihydro quinoline-3-carboxylic acid" in the key residues Ser79 as well as DG1 in the DNA groove. 44The synthetic compounds' docking results include the binding affinity Score and Root Mean Square Deviation (RMSD).The following table lists the ligand interactions with the active site residues, including hydrogen bonding and hydrophobic interactions (Table 4).

In-silico ADME assessment
The synthesized compounds were put through an in-silico investigation to determine their pharmacokinetic characteristics using Swiss ADME 62 .The investigated compounds demonstrated high GI absorption ability except for compound 7e.Moreover, the tested hybrids show low Blood Brain Barrier (BBB) permeability (Table 5).Cell membrane penetrability reflected by the log P values, range from 2.77 to 3.91, these values are < 5 which reflects marked cell membranes tolerability 63 .Following "rule of five" for Lipinski, all compounds' molecular weights were less than 500 except for compound 7e which was slightly higher than 500 recording value of 531.99.In addition, Hydrogen-Bond Donors (HBD) were 2 and Hydrogen-Bond Acceptors (HBA) were 4 for all tested compounds, with rotatable bonds ranging from 4 to 7. The bioavailability score was 0.55 for all compounds except 7e which recorded the lowest bioavailability score 0.17, this might be attributed to its high molecular weight.All screened compounds had promising ADME data that revealed compliance with the Lipinski's rule except for compound 7e that had two Lipinski's violations with high molecular weight and low bioavailability score which affected its GI absorption probabilities (Table 6).

Structural activity relationship
The main objective of the installation of new hybrids containing coumarin and heteroarenes bearing two, three and four fused cyclic rings is to optimize and reach the best activity depending on the synergistic effect of the utilized precursors.Notably, the acridine and neocryptolepine pharmaceutical cores have quinoline motif fused with benzyl as showed in acridine or indole in case of neocryptolepine.Relying on the and from the reported IC 50 values in Table 2, structure activity relationship of the synthesized hybrids was studied.Table 1 emphasized the presence of anticancer activity of these hybrids comparing to their starting core used as positive control and revealed the synergistic effect of these hybrids.In light of the afforded results of the synthesized hybrids against MDA-MBA-231 cancer cell line, it is noteworthy that hybrid 7c containing coumarin and quinoline cores as two fused rings with presence of three carbon spacers of quinolone core showed the best activity with IC 50 :50.96µM and SI:2.2 folds among of their relatives presenting in hybrids 7a-e.Furthermore, it is noteworthy that hybrid 7c containing two fused rings with propyl spacer at C-4 of quinoline core had higher activity than tetra-and tri cyclic ring belonging to neocryptolepine and acridine cores with the same spacer attached on C-11 and C-9 resembled to 13c and 10c respectively.On the other hand, hybrid 13c showed antiproliferative activity against HepG-2 cell line compared to their relative hybrids 10c and 7c.In addition to, IC 50 of A549 cell line illustrated that 13c have the highest potency than 7c and 10c.Furthermore, hybrid 7d showed the best activity against CaCO-2 cell line which have phenylene diamine as spacer with two fused rings.That illustrated that length of spacer influences the antiproliferative activity.In addition 10b and 13b, which contained two carbons spacer with tri and tetra fused rings, showed higher antibacterial and antifungal activity against S.aureus, s.mutans, E.coli and C.albicans.

Conclusions
In the current study, hybrids of coumarin-quinolines 7a-e, coumarin-acridines 10b,c and coumarin-neocryptolepines 13b,c were synthesized and evaluated for their antiproliferative and antimicrobial activities.Hybrids 7c and 7d were proved to be the most potent as an antiproliferative agents, showing inhibitory activity against MDA-MB-231 and CaCo-2 with IC 50 of 50.96 and 51.95 µM respectively.Furthermore, Hybrids 10b and 13b showed higher antibacterial activity against S.aureus, S.mutans and E. coli with MIC from 3.2 to 15 µM compared to their corresponding derivatives 7a-e and 10c and 13c.as well as potent inhibitory activity against Candida albicans with MIC 0.0011 to 0.12 µM.Molecular docking study disclosed that the planar di or tri ring aromatic system showed hydrophobic interactions at the binding site of action mostly with the amino acids residues Asp78, Asp83, Ser79 and DG1, DC4, and DG5 residues at the DNA-minor groove.These interactions are consistent with the reported interactions by the co-crystallized ligand of topoisomerase protein in the key residue Ser79 and in     DG1 of the DNA groove.All screened compounds showed promising ADME data following Lipinski's rule except for compound 7e that had two Lipinski's violations with high molecular weight and low bioavailability score which affected its GI absorption probabilities.Overall, the results of this study support the possibility of using these coumarin hybrids as promising anticancer and antimicrobial agents for further in vivo animal model study.

Figure 1 .
Figure 1.Design of coumarin N-heterocyclic hybrids as potent antiproliferative and antimicrobial agents.

Figure 3 .
Figure 3. MIC values of the synthesized coumarin hybrid compounds against Staphylococcus aureus and Candida albicans.

Figure 4 .
Figure 4. MIC values of the synthesized coumarin hybrid compounds against Pseudomonas aeruginosa Streptococcus mutans and E. coli.

Figure 5 .
Figure 5. (A-D) Docking results of the synthesized compounds, best scoring compounds' interactions 7b (in green sticks) and 7e (in cyan sticks) inside the active site of topoisomerase 2 with H-bonds in green, Pi-Pi stacking in pink, pi-H bonds in light pink and pi anion bonds in orange color.

Table 1 .
IC 50 values of measured coumarin hybrid compounds compared to DOX as reference drug.

Table 2 .
Selective index values of measured coumarin hybrid compounds.

Table 3 .
MIC values of the synthesized coumarin hybrids against Staphylococcus aureus, Candida albicans, E. coli, Streptococcus mutans and Pseudomonas aeruginosa.

Table 4 .
Receptor binding affinity, RMSD values and residues involved in the interaction at the receptor active site.